Hydrogen sulfide is involved in maintaining ion homeostasis via regulating plasma membrane Na+/H+ antiporter system in the hydrogen peroxide-dependent manner in salt-stress Arabidopsis thaliana root

Hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) function as the signaling molecules in plants responding to salt stresses. The present study presents a signaling network involving H₂S and H₂O₂ in salt resistance pathway of the Arabidopsis root. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage (EL) and Na⁺/K⁺ ratio under salt stress. The treatment of H₂S donors sodium hydrosulfide (NaHS) enhanced the salt tolerance by maintaining a lower Na⁺/K⁺ ratio. In addition, the inhibition of root growth under salt stress was removed by H₂S. Further studies indicated that H₂O₂ was involved in H₂S-induced salt tolerance pathway. H₂S induced the production of the endogenous H₂O₂ via regulating the activities of glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) NADPH oxidase, with the treatment with dimethylthiourea (DMTU, an ROS scavenger), diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor), or glycerol (G6PDH inhibitor) removing the effect of H₂S. Treatment with amiloride (an inhibitor of PM Na⁺/H⁺ antiporter) and vanadate (an inhibitor of PM H⁺-ATPase) also inhibited the activity of H₂S on Na⁺/K⁺ ratio. Through an analysis of quantitative real-time polymerase chain reaction and Western blot, we found that H₂S promoted the genes expression and the phosphorylation level of PM H⁺-ATPase and Na⁺/H⁺ antiporter protein level. However, when the endogenous H₂O₂ level was inhibited by DPI or DMTU, the effect of H₂S on the PM Na⁺/H⁺ antiporter system was removed. Taken together, H₂S maintains ion homeostasis in the H₂O₂-dependent manner in salt-stress Arabidopsis root.     

Withaminimin,a withanolide from Physalis minima

The structure of withaminimin, a new ergostane-type steroid from Physalis minima, was established by spectral analysis (¹H and ¹³C NMR, MS) and chemical transformations, as (20S,22R)-15α-acetoxy-5α,6β,14α-trihydroxy-1-oxowitha-2,16,24-trienolide. An unusual MH₂O⁺ quasi-molecular ion was observed in the chemical ionization mass spectrum of the natural product.     

Analysis of Modified Deoxynucleosides by Electrospray Ionization Mass SPECTROMETRY

Abstract

Electrospray mass spectra for selected modified deoxynucleosides and deoxynucleoside monophosphates have been determined. Protonated molecular ions are abundant in the positive ion spectrum, while (M-H) appears in the negative ion spectrum. However, fragment ion intensities are usually low in both spectra. Conditions which promote collision-induced dissociation within the electrospray source facilitate fragment ion formation, and the intensity of BH₂ ⁺ and S⁺ (positive ion spectrum) and (M-BH) and B (negative ion spectrum) are enhanced by increasing the skimmer cone voltage. MH⁺ was detected with as little as 3 pmol of deoxynucleoside, and the protonated molecular ion intensity is linear with respect to analyte concentration over two orders of magnitude.     

Temperature Dependence of Voltage-gated H+ Currents in Human Neutrophils,Rat Alveolar Epithelial Cells,and Mammalian Phagocytes

H⁺ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42°C. Effects of temperature on gating kinetics were distinguished from effects on the H⁺ current amplitude. The activation and deactivation of H⁺ currents were both highly temperature sensitive, with a Q ₁₀ of 6–9 (activation energy, E _a, ≈ 30–38 kcal/mol), greater than for most other ion channels. The similarity of E _a for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H⁺ currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant (τ_act) had similarly high Q ₁₀. These results could be explained if H⁺ channels were composed of several subunits, each of which undergoes a single rate-determining gating transition. H⁺ current gating in all mammalian cells studied had similarly strong temperature dependences. The H⁺ conductance increased markedly with temperature, with Q ₁₀ ≥ 2 in whole-cell experiments. In excised patches where depletion would affect the measurement less, the Q ₁₀ was 2.8 at >20°C and 5.3 at <20°C. This temperature sensitivity is much greater than for most other ion channels and for H⁺ conduction in aqueous solution, but is in the range reported for H⁺ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H⁺ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large E _a of permeation intrinsically limits the conductance of this channel, and appears inconsistent with the channel being a water-filled pore. At physiological temperature, H⁺ channels provide mammalian cells with an enormous capacity for proton extrusion.     

A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca²⁺ and K⁺ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K⁺ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H₂O₂. We show that H₂O₂ rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H₂O₂ and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H₂O₂-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys¹⁶⁸ located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H₂O₂. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys¹⁶⁸ as a critical target for H₂O₂, and implicate ROS-mediated control of the K⁺ channel in regulating mineral nutrient partitioning within the plant.     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

Effects of H₂O₂ on electrical membrane properties of skeletal myotubes

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K⁺ (K_IR) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba²⁺ and Cs⁺. The block of the outward tail current contributing to K_IR deactivation can explain the shorter latency for spike initiation. We propose that the K_IR current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.     

ADP Is a Competitive Inhibitor of ATP-Dependent H Transport in Microsomal Membranes from Zea mays L. Coleoptiles

An anion-sensitive ATP-dependent H⁺ transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H⁺ transport observed: strict dependence on Cl⁻; inhibition by SO₄²⁻ and NO₃⁻; insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl₂ (H⁺/Ca²⁺ antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl₂ with a K_m value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H⁺ transport significantly. However, ADP was a potent competitive inhibitor with a K_i value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure.     

The effect of H<Subscript>3</Subscript>O<Superscript>+</Superscript> on the membrane morphology and hydrogen bonding of a phospholipid bilayer

At the 2017 meeting of the Australian Society for Biophysics, we presented the combined results from two recent studies showing how hydronium ions (H₃O⁺) modulate the structure and ion permeability of phospholipid bilayers. In the first study, the impact of H₃O⁺ on lipid packing had been identified using tethered bilayer lipid membranes in conjunction with electrical impedance spectroscopy and neutron reflectometry. The increased presence of H₃O⁺ (i.e. lower pH) led to a significant reduction in membrane conductivity and increased membrane thickness. A first-order explanation for the effect was assigned to alterations in the steric packing of the membrane lipids. Changes in packing were described by a critical packing parameter (CPP) related to the interfacial area and volume and shape of the membrane lipids. We proposed that increasing the concentraton of H₃O⁺ resulted in stronger hydrogen bonding between the phosphate oxygens at the water–lipid interface leading to a reduced area per lipid and slightly increased membrane thickness. At the meeting, a molecular model for these pH effects based on the result of our second study was presented. Multiple μs-long, unrestrained molecular dynamic (MD) simulations of a phosphatidylcholine lipid bilayer were carried out and showed a concentration dependent reduction in the area per lipid and an increase in bilayer thickness, in agreement with experimental data. Further, H₃O⁺ preferentially accumulated at the water–lipid interface, suggesting the localised pH at the membrane surface is much lower than the bulk bathing solution. Another significant finding was that the hydrogen bonds formed by H₃O⁺ ions with lipid headgroup oxygens are, on average, shorter in length and longer-lived than the ones formed in bulk water. In addition, the H₃O⁺ ions resided for longer periods in association with the carbonyl oxygens than with either phosphate oxygen in lipids. In summary, the MD simulations support a model where the hydrogen bonding capacity of H₃O⁺ for carbonyl and phosphate oxygens is the origin of the pH-induced changes in lipid packing in phospholipid membranes. These molecular-level studies are an important step towards a better understanding of the effect of pH on biological membranes.     

Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase

Mitoxantrone (MH₂X), an anthraquinone-type anti-cancer agent used clinically in the treatment of human malignancies, is oxidatively activated by the peroxidase/H₂O₂ enzyme system. In contrast to the enzymatic mechanisms of drug oxidation, the chemical transformations of MH₂X are not well described. In this study, MH₂X metabolites, produced by the horseradish, lacto- or lignin peroxidase (respectively HRP, LPO and LIP)/H₂O₂ system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar mitoxantrone/H₂O₂ ratio, the efficacy of the enzyme-catalyzed oxidation of mitoxantrone decreased in the following order: LPO > HRP > LIP, which accorded with the decreasing size of the substrate access channel in the enzyme panel examined. In all cases, the central drug oxidation product was the redox-active cyclic metabolite, hexahydronaphtho-[2,3-f]-quinoxaline-7,12-dione (MH₂), previously identified in the urine of mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH₂ side-chains occurred, yielding the mono- and dicarboxylic acid derivatives respectively. Based on the available data a further MH₂ derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H₂O₂ concentrations, these novel MH₂ derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH₂ chromophore due to an oxidative cleavage of the 9,10-anthracenedione moiety. The novel metabolites extend the known sequence of peroxidase-induced mitoxantrone metabolism, and may contribute to the cytotoxic effects of the drug in vivo. Based on the structural features of the proposed MH₂ oxidation products we elaborate on various biochemical mechanisms, which extend the understanding of mitoxantrone’s pharmaceutical action and its clinical effectiveness with a particular focus on peroxidase-expressing solid tumors, such as breast carcinoma.     

Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis

Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na⁺/K⁺ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H₂O₂) generated in the presence of heme in the Na⁺/K⁺ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H₂O₂ in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na⁺/K⁺ ATPase is through the generation of H₂O₂, we measured enzyme activity using increasing concentrations of H₂O₂ and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H₂O₂. To investigate the role of PKC in this signaling pathway, we observed the production of H₂O₂ in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H₂O₂. Furthermore, we found that PKC activity is increased in the presence of H₂O₂, and that in the presence of calphostin C, H₂O₂ is unable to activate the Na⁺/K⁺ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na⁺/K⁺ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na⁺/K⁺ ATPase by H₂O₂ opens new possibilities for understanding the signaling pathways of this parasite.     

Electrospray identification of new polyoxochromate species

Electrospray ionization mass spectrometry (ESI MS) has been conducted on the ammonium and alkali metal (A=Li⁺, Na⁺ and K⁺) dichromate systems. A large number of previously unknown polyoxochromate species have been characterized. Major series that have been identified include [A_x+1H_xCr^VI_xO_4x]⁺ (Li⁺, x=1-5; Na⁺, x=1-7; K⁺, x=1-4) and [A_2x−1Cr^VI_xO_4x−1]⁺ (Li⁺, x=2, 3; Na⁺, x=2-4; K⁺, x=2, 3) in the alkali metal dichromate systems, and [HCr^VI_xO_3x+1]⁻ (x=1-5) in the ammonium dichromate system. Several series also contain mixed oxidation state species, ranging from Cr(V) to Cr(II) in conjunction with Cr(VI), which is consistent with the ease of reduction of Cr(VI). Negative ion ESI MS spectra clearly demonstrate the existence of [HCrO₄]⁻ as the most abundant ion at −20 V, suggesting that its existence in solution is not just hypothetical, as was previously thought. The polymerization units for the series observed include {AHCrO₄}, {A₂CrO₄} and {CrO₃}, with the latter prominent in the alkali metal systems. This presumably arises from the fragmentation of dichromate, A₂Cr₂O₇→{A₂CrO₄}+{CrO₃}. Moreover, the ESI MS of the dichromate compounds have illustrated that the preservation of tetrahedral stereochemistry is of paramount importance for these systems, which leads to only limited polymerization compared to the related molybdate and tungstate systems.     

The use of a sulfide ion selective electrode to study O-acetylserine sulfhydrylase from germinating rapeseed

O-Acetylserine sulfhydrylase was partially purified from germinating seeds of Rape (Brassica napus L. var Target), and a sulfide ion specific electrode used to study the properties of this enzyme. It was demonstrated that the sensitivity of the electrode to sulfide ion concentration was not affected by pH, or by various concentrations of cysteine, sulfate, sulfite, and acetate.Kinetic studies showed that the K_m for O-acetyl-l-serine was 1.74 × 10^?6m, while the K_m for sulfide was 4.3 × 10^?4m. The implications of the data, together with the methods used to obtain them, are discussed.     

Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli

Hydrogen peroxide (H₂O₂, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H₂O₂ presents several questions. First, the lethal H₂O₂ concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H₂O₂-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H₂O₂ toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H₂O₂ antimicrobial action, others instead report alleviation. To investigate this “NO paradox,” we treated Escherichia coli with various concentrations of H₂O₂-alone or H₂O₂+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H₂O₂ treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H₂O₂ toxicity by blocking H₂O₂ scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H₂O₂-alone or H₂O₂+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H₂O₂-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H₂O₂ concentrations is transiently alleviated by NO, reproducing the “NO paradox.” We conclude that NO potentiates H₂O₂ toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H₂O₂ concentrations, while transiently suppressing mode-two killing by immediately lethal high H₂O₂ concentrations.     

A Highly Temperature-sensitive Proton Current in Mouse Bone Marrow–derived Mast Cells

Proton (H⁺) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q₁₀ of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q₁₀ was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K⁺, Cl⁻, and Na⁺), but depended on the pH gradient, suggesting that H⁺ (equivalents) is a major ion species carrying the current. The H⁺ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H⁺ current was reversibly inhibited by Zn²⁺ but not by bafilomycin A₁, an inhibitor for a vacuolar type H⁺-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn²⁺, and depletion of extracellular K⁺, but not by bafilomycin A₁. These results suggest that the H⁺ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H⁺ conductance.     

Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H⁺-PPase) from oat (Avena sativa L.) roots to changes in Mg²⁺ and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H⁺-PPase is Mg₂PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg²⁺ and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H⁺-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H⁺-PPase. It is demonstrated that models incorporating Mg₂PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H⁺-PPase. Calculations of the likely concentrations of Mg₂PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H⁺-PPase would be about 70% saturated in vivo.     

Upregulation of Na/H exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O₂ deficiency glycolytic degradation of glucose leads to formation of lactate and H⁺, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H⁺ export. The present study explored whether AMPK influences Na⁺/H⁺ exchanger (NHE) activity and/or Na⁺-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH_i) was estimated utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺-dependent re-alkalinization (ΔpH_i) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH_i and ΔpH_i in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pH_i and ΔpH_i was blunted in the presence of the Na⁺/H⁺ exchanger inhibitor cariporide (10 μM), but not by the H⁺ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.     

Kinetics and mechanisms of the CO2 and SO2 uptake by coordinate ion, cis-[Cr(C2O4)(L-L)(OH2)2] {(L-L) = methyl 3-amino-2,3-dideoxy-α-d-arabino-hexopyranoside} as studied by stopped-flow spectrophotometry

The kinetics of CO₂ and SO₂ uptake by a coordinate ion, cis-[Cr(C₂O₄)(L-L)(OH₂)₂]⁺, where L-L stands for a bidentate sugar ligand, methyl 3-amino-2,3-dideoxy-α-d-arabino-hexopyranoside has been studied, over temperature ranges of 5 - 25 and 5 - 20 °C for CO₂ and SO₂, respectively. Investigations were carried out using stopped-flow spectrophotometry in the range of 340-700 nm. Results of the kinetic measurements obtained for both gases were compared. The kinetics and mechanisms of the reactions were suggested and ΔH^‡ values for both processes were determined.